Periodontitis is a disease of the subgingival crevice that leads to soft tissue degeneration and tooth loss. The bacterium Aggregatibacter actinomycetemcomitans (Aa) has been implicated in localized aggressive periodontitis, in several adult periodontal disorders and in life-threatening systemic infections. We are interested in determining how Aa is a successful periodontal pathogen. Our efforts have focused on defining novel regulatory pathways in Aa that accurately control expression of the virulence factors needed to thrive in different conditions, with the long term goal of developing therapeutic protocols to block the synthesis of coordinately regulated virulence proteins. Members of the sirtuin (Sir2-like) family of proteins are found in all three domains of life. These enzymes, which are NAD+-dependent protein deacetylases, play an important role in eukaryotic and archaeal transcriptional regulation by removing acetyl groups from lysines on various DNA-binding proteins. The bacterial Sir2 homologue is called CobB. In Salmonella enterica, this deacetylase has been shown to regulate the enzymatic activity of several enzymes in intermediary metabolism. Recently, three groups determined that ~90 proteins, including two transcription factors, are acetylated in E. coli and that S. enterica has 191 acetylated proteins, including nine transcription factors. However, the effect of acetylation on the activities of the transcription proteins was not tested. Given that the Sir2 deacetylase is important in controlling eukaryotic and archaeal transcription and that transcription factors can be acetylated in bacteria, we postulated that lysine acetylation of transcriptional regulatory proteins could play an important role in shaping the RNA expression patterns in bacteria, something that has not been shown in any bacterium. Thus, we made a deletion mutant of the Aa cobB deacetylase gene. Microarray analysis of RNA from this mutant showed that the alteration of acetylation levels in Aa leads to the mis-regulation of forty-eight RNAs, suggesting, but not proving, that lysine-acetylation of transcription factors will be critical for transcriptional regulation in bacteria. In this proposal, we will test this hypothesis. First, mass spectrometry will be used to identify the Aa acetylome, including its lysine-acetylated transcription factors. Subsequently, we will construct a set of site-specific mutants, which mimic an acetylated lysine or a deacetylated lysine, in the acetylated- lysine codon for each of the genes encoding any of the acetylated transcription factors found. Finally, microarray studies with the mutants will reveal the global RNA expression profiles of each acetylated mutant, and its corresponding deacetylated mutant, relative to wild type cells. Finding different patterns of transcription among these strains would prove, for the first time in any bacterium, that lysine-acetylation of transcriptional regulatory proteins plays a key role in modulating bacterial RNA expression. Importantly, since CobB is an NAD+-dependent deacetylase, our results would also suggest that there is a possible new connection between physiology (NAD levels) and transcriptional regulation (acetylation) in bacteria. PUBLIC HEALTH RELEVANCE: Periodontitis, an inflammatory disease of tissues in the subgingival crevice, is associated with a dramatic shift in the subgingival microflora towards gram negative bacteria. Certain forms of this disease are caused by the facultative bacterium Aggregatibacter actinomycetemcomitans which is also considered a risk factor in cardiovascular diseases. It is important to characterize new regulatory mechanisms used by this organism, such as acetylation of transcription factors in response to physiologic changes, since such pathways may serve as novel targets for future therapeutic intervention.